Authors: Lin, Tzu-Chen
Title: Optochemical control of DNA methylation erasers and readers enables kinetic insights into their domain-dependent interplay
Language (ISO): en
Abstract: 5-Methylcytosine (5mC) is a central epigenetic mark of mammalian DNA. It mainly occurs in cytosine-guanine (CpG) dinucleotides and is recognized competitively by methyl-CpG binding domain (MBD) proteins and ten-eleven-translocation (TET) dioxygenases, which act as methylation readers and erasers to mediate regulatory chromatin crosstalk and epigenome editing, respectively. The dynamic reader-eraser interplay at their common substrate is therefore highly regulated for a coherent transcriptional program. However, mechanistic insights of their interplay are hampered by a lack of suitable methodology to control their activities in living cells. This work employs light-activatable human TET1 and MBD1 to enable precise temporal control of enzymatic oxidation activity or substrate recognition. Light activation is achieved by genetic encoding of a photocaged serine that can be co-translationally incorporated at critical protein sited in mammalian cells. On the one hand, monitoring the TET1-catalyzed 5mC oxidation kinetics in vivo reveals a multifaceted domain-dependent modulation by MBD1. While the MBD domain of MBD1 negatively regulates TET1 oxidation kinetics and dominates the interplay by competing for the 5mC substrates, the third Cys-x-x-Cys (CXXC3) domain of MBD1 contrarily modulates TET1 activity by binding to nonmethylated CpGs. Intriguingly, the transcriptional repressor domain (TRD) does not influence 5mC oxidation kinetics by TET1. On the other hand, studies with light-activatable MBD1 indicate a domain-dependency of cellular mCpG binding kinetics. Depriving the nonmethylated CpG affinity of the CXXC3 domain enhances binding kinetics, whereas the absence of the TRD domain results in decreased binding kinetics. Moreover, the light-activatable MBD1 can further unveil the mechanism of MBD1-TET1 interplay by uncoupling the process from prior binding events of MBD1. Collectively, this work enables first kinetic insights into the domain-dependent interplay of methylation readers and erasers in the natural chromatin context and provides novel tools to unravel the dynamic chromatin regulation program.
Subject Headings: DNA methylation
MBD protein
TET dioxygenase
Optochemical control
Subject Headings (RSWK): DNA
Issue Date: 2022
Appears in Collections:Chemische Biologie

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